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J. Chem. Inf. Model. 2006, 46, 2692-2697
The Importance of Local Chemical Structure for Chemical Metabolism by Human Uridine 5′-Diphosphate-Glucuronosyltransferase Michael J. Sorich,*,† Ross A. McKinnon,† John O. Miners,‡ and Paul A. Smith‡ Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia, Frome Rd, Adelaide, SA 5000, Australia, and Department of Clinical Pharmacology, Flinders University, Bedford Park, SA 5042, Australia Received June 20, 2006
The uridine 5′-diphosphate- (UDP-)glucuronosyltransferase (UGT) family of enzymes catalyzes the conjugation of chemicals containing a suitable nucleophilic atom with glucuronic acid. Despite the importance of glucuronidation as an elimination and detoxification mechanism for drugs, environmental chemicals, and endogenous compounds, the structural features of substrates that confer isoform selectivity are poorly understood. The relationship between the local molecular structure of nucleophilic atoms of chemicals and the ability of UGT isoforms to glucuronidate the nucleophilic atoms was investigated here. The proximity of an aromatic ring to the nucleophilic atom was highly associated with a greater likelihood of glucuronidation by most UGT isoforms. Similarly, most UGT isoforms were found to have a statistically significant preference for oxygen over nitrogen as the nucleophilic atom. The converse was established only for UGT1A4. Naı¨ve Bayes models were trained to predict the site of glucuronidation for eight UGT isoforms on the basis of the partial charge and Fukui function of the nucleophilic atom and whether an aromatic ring was attached to the nucleophilic atom. On average, the cross-validated sensitivity and specificity of the models were approximately 75-80%. For all but UGT2B7, the area under the receiver operating characteristics curve of the model was greater than 0.8, indicating strong predictive ability. A chemical diversity analysis of the currently available data indicates bias toward chemicals containing phenolic groups, and it is likely that the availability of chemical data sets with greater diversity will facilitate further insights into the structural features of substrates that confer enzyme selectivity. INTRODUCTION
Humans are exposed on a daily basis to a diverse array of foreign chemicals (xenobiotics), including pharmaceutical, environmental, and industrial chemicals. The detoxification of xenobiotics within the human body relies heavily on chemical modification (metabolism) by a relatively small number of enzymes.1 Metabolism promotes excretion of the chemical via the kidneys or bile and, generally, reduces pharmacological and toxicological activity. While most enzymes only interact with a small set of structurally similar chemicals, some chemical detoxification enzymes have evolved the ability to metabolize chemicals of highly divergent sizes, shapes, flexibilities, complexities, and hydrophobicities (e.g., Figure 1).1 However, the structural features of substrates that confer enzyme selectivity are frequently poorly understood. The uridine 5′-diphosphate- (UDP-)glucuronosyltransferase (UGT) family of enzymes comprises 15 functional “isoforms” that catalyze the conjugation of chemicals containing a suitable nucleophilic atom (mainly, aliphatic or aromatic hydroxyl, carboxyl, or amino groups) with glucuronic acid, according to a second-order nucleophilic substitution mechanism.2-4 Glucuronidation serves as a * Corresponding author tel.: +61 8 83021230; fax: +61 8 83021087; e-mail:
[email protected]. † University of South Australia. ‡ Flinders University.
clearance mechanism for drugs from all therapeutic classes, including some with a narrow therapeutic index.2-10 In addition, glucuronidation acts as an elimination pathway for numerous other endogenous, dietary, and environmental chemicals.4 A single UGT isoform can catalyze the glucuronidation of multiple “acceptor” sites that may be present within a single compound,5,6,8,10 suggestive of multiple substrate binding modes. Furthermore, the kinetic modeling of xenobiotic glucuronidation is consistent with multiple substrate binding sites within the UGT active site.11,12 UGT is a membrane-bound protein with a poorly defined threedimensional structure (the most similar protein with a solved high-resolution crystal structure shares approximately 15% sequence identity). Consequently, insights into the molecular mechanism underlying substrate recognition are predominantly based on small molecule modeling.3,13-23 Previously, in silico models were made to predict substrates and nonsubstrates of human UGT isoforms (“reaction phenotyping”).22 It was demonstrated that both 2D molecular descriptors and quantum-chemical molecular descriptors were useful for discriminating substrates from nonsubstrates.20,22 Multiple pharmacophore perception studies gave insight into the chemical features associated with substrates and nonsubstrates of human UGT isoforms.21 The individual pharmacophores differed between isoforms but, generally, represented relatively simple structural and chemical features.21 Furthermore, the pharmacophores suggested that the environ-
10.1021/ci600248e CCC: $33.50 © 2006 American Chemical Society Published on Web 09/22/2006
LOCALIZED MOLECULAR RECOGNITION
Figure 1. Examples of structurally diverse chemicals that bind to UGT1A1.
ment immediately adjacent to the nucleophilic site of conjugation may be an important determinant of metabolism by a particular UGT.21 However, because of a lack of data on actual sites on glucuronidation on the substrates, this was not able to be confirmed. To gain a greater insight into the mechanisms underlying glucuronidation, this study seeks to understand the features of nucleophilic atoms of chemicals that effect whether a reaction occurs at specific nucleophilic atoms or not. MATERIALS AND METHODS
Data Sets. Potential sites of glucuronidation on a chemical include the following nucleophilic atoms: oxygen in a hydroxyl group, singly bonded oxygen in a carboxylic acid group, nitrogen in an amine group or in a heterocyclic ring, and sulfur in a thiol group. For each UGT isoform, a data set was collated from the literature on nucleophilic atoms which are glucuronidated and nucleophilic atoms which are not glucuronidated (see the Supporting Information). A nucleophilic atom is deemed to be glucuronidated if (1) the specific position of the glucuronide was experimentally determined following an assay of the chemical in an in vitro recombinant cell system expressing a single UGT isoform or (2) the chemical contains only one nucleophilic atom and the chemical was determined to be glucuronidated in an in vitro recombinant cell system expressing a single UGT
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isoform. Similarly, a nucleophilic atom was deemed not to be glucuronidated if (1) the glucuronide could not be detected following an assay of the chemical in an in vitro recombinant cell system expressing a single UGT isoform or (2) the chemical was found not to be glucuronidated at all in an in vitro recombinant cell system expressing a single UGT isoform. Hierarchical Clustering. For each UGT isoform, the nucleophilic atoms in the data set were assigned to one or more functional group types: phenolic (Ar-OH), aliphatic hydroxyl (Al-OH), carboxylic acid (COOH), amine adjacent to aromatic (Ar-NRx) aliphatic amine (Al-NRx), primary amine (NR1), secondary amine (NR2), tertiary amine (NR3), heterocyclic aromatic nitrogen (n), and thiol (SH). For each functional group type with more than 10 known nucleophilic atoms assigned for a particular UGT isoform, the percentage of nucleophilic atoms glucuronidated was calculated. If insufficient data existed, the percentage was labeled “NA”. To display the similarity of nucleophilic atom preference between UGT isoforms and between functional group types, the percentage of each functional group type glucuronidated by individual UGT isoforms was hierarchically clustered using the Euclidean distance with “NA” data masked.24 The percentage of each local structure glucuronidated by each UGT isoform is visually represented as a color map, using linear interpolation between the dark blue and light blue representing 100% glucuronidation and 0% glucuronidation, respectively. Statistical Tests of Local Chemical Properties. When the R statistical environment was used,25 the Fisher exact test26 was used to determine the statistical significance of the effect of two local features on the likelihood of nucleophilic atom glucuronidation. For each UGT isoform, the count of glucuronidated and nonglucuronidated nucleophilic atoms was determined for (1) nucleophilic atoms attached to an aromatic ring versus those not attached to an aromatic ring and (2) nucleophilic nitrogen atoms (e.g., all amines and aromatic nitrogens) versus nucleophilic oxygen atoms (e.g., all hydroxyls and carboxylic acids). The partial charge and Fukui function were calculated using an in-house implementation of the electronegativity equalization method.27 The nonparametric Mann-Whitney28 test implemented in R25 was used to determine whether the partial charge and Fukui function of the nucleophilic atom were useful for discriminating glucuronidation reactivity. Naı1ve Bayes Models. To determine the importance of the local structure for the prediction of glucuronidation of the nucleophilic atom, a naı¨ve Bayes model29 (implemented in the Orange data mining software30) was trained for each UGT isoform for which at least 20 examples existed of nucleophilic atoms that are and are not glucuronidated. Naı¨ve Bayes classification was chosen for its simplicity and ability to handle unbalanced data sets.29 Three explanatory variables were used: a Boolean variable indicating whether an aromatic ring was attached to the nucleophilic atom, the partial charge of the nucleophilic atom, and the atom-centered Fukui function. To produce models with similar classification performance for glucuronidated and nonglucuronidated nucleophilic atoms, weighting of the two groups was set to counteract the imbalance in the training set sizes of the two groups. The predictive ability of the models was assessed using 10-fold cross-validation with stratification of the
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Figure 2. Clustering and color representation of glucuronidation frequency of different nucleophile types by 14 UGT isoforms (1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2B4, 2B7, 2B15, 2B17, and 2B28). The nucleophile types displayed include the following functional groups: phenolic (Ar-OH), aliphatic hydroxyl (Al-OH), carboxylic acid (COOH), amine adjacent to aromatic (Ar-NRx), aliphatic amine (Al-NRx), primary amine (NR1), tertiary amine (NR3), and aromatic nitrogen (n).
samples on the basis of class.31 This assessment method is generally equivalent to using a random test set consisting of 10% of the data, but with the process of selecting and assessing the test set repeated 10 times and averaged (see ref 31 for a review on cross-validation). As a result, the test set performance is not so sensitive to the selection of the test set. Sensitivity, specificity, and the area under the receiver operating characteristic curve (AROC)32 were subsequently used to summarize the predictive ability of the model. Comparison of Chemical Libraries. The set of approximately 500 chemicals that have been tested for glucuronidation by individual UGT isoforms was compared against the NCI diversity set33 of approximately 2000 chemicals and the 3D Pharmaceutical Structure Database (http://www.ps.toyaku.ac.jp/dobashi/) of approximately 1000 drugs. The number of chemicals that contained each of the functional group types was ascertained using SMARTS substructure searching implemented in the Frowns toolkit (http://frowns.sourceforge.net/). The frequency of a functional group was defined as the number of chemicals that contain the functional group divided by the total number of chemicals in the specific chemical library. RESULTS AND DISCUSSION
The color map in Figure 2 displays the glucuronidation frequency of nucleophilic atoms with differing local structures and substitutions. The classifications are based on standard functional-group types, as this is an intuitive and informative way to represent both the local electronic and steric environment of the nucleophilic atom. It is immediately
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apparent from the color map that differences exist in the susceptibility of different nucleophile types to glucuronidation, indicating that the local environment of the nucleophilic atom has an important influence on the potential for glucuronidation. One striking feature of Figure 2 is the difference in susceptibility to glucuronidation between aliphatic (Al-OH and Al-NRx) and aromatic (Ar-OH and Ar-NRx) nucleophilic groups. For almost all UGT isoforms, it appears that an aromatic ring attached to the nucleophilic atom increases, often dramatically, the susceptibility of the nucleophilic atom to glucuronidation. This observation was subsequently statistically assessed using the Fisher exact test and MannWhitney test (Table 1). For most of the isoforms for which sufficient data exist, the differences between nucleophilic atoms attached to an aromatic ring and those not attached to an aromatic ring were highly statistically significant. Differences were nonsignificant only for UGT1A4 and UGT2B7. It is possible that the aromatic ring increases the likelihood of glucuronidation at the nucleophilic atom by enhancing the percentage of productive substrate-binding events within the active site, perhaps by forming π-stacking interactions with aromatic ring(s) in the active site.34 Such an interaction may align the nucleophilic atom more appropriately for reaction with the UDP-glucuronic acid cofactor and augment the reaction rate by increasing the frequency of successful bond forming or by decreasing the entropy of bond formation.34 It is also possible that the aromatic ring may increase the reaction rate by electronically stabilizing the reaction intermediate. Figure 2 also indicates that, within analogous environments, nucleophilic oxygen atoms may be preferred for glucuronidation over nucleophilic nitrogen atoms. This observation was also verified statistically by determining the probability that differences in susceptibility to glucuronidation between nucleophilic nitrogen and oxygen atoms can be accounted for by chance (Table 1). For many isoforms, the probability of these differences occurring by chance is extremely unlikely (p < 0.001). UGT1A4 is the exception to the rule, showing a preference for glucuronidation of nucleophilic nitrogens over oxygens. For all UGT isoforms analyzed, the partial charge and the Fukui function were on average more negative and higher in nucleophilic atoms that are glucuronidated, respectively. In almost all cases, the differences were highly statistically significant. The association of negative charge and the Fukui function with glucuronidation is probably because they indicate greater nucleophilicity. The Fukui function indicates how incoming or outgoing electrons are redistributed in various regions of the molecule. As such, the atom-condensed Fukui function (the Fukui function integrated over the region attributed to an atom) is useful as an indicator of the relative atomic susceptibility to electrophilic or nucleophilic attack.35-37 Naı¨ve Bayes classifiers were developed in order to estimate how well the glucuronidation of nucleophilic atoms could be predicted on the basis of the local environment of the nucleophilic atom. Such models predict the glucuronidation of nucleophilic atoms by each UGT isoform using only three simple descriptors of the nucleophilic atom’s local structure. The percentage of glucuronidated and nonglucuronidated nucleophilic atoms predicted correctly, as determined by 10fold cross-validation, is generally high (Table 1). The median
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Table 1. Data Set Size, Association with Descriptors, and Multivariate Model Results for each UGT isoform
number of nucleophilic sites isoform UGT1A1 UGT1A3 UGT1A4 UGT1A6 UGT1A8 UGT1A9 UGT1A10 UGT2B7
glucuronidated 36 72 54 54 51 81 47 59
not glucuronidated 253 104 201 267 45 184 183 150
p value (Fisher exact or Mann-Whitney test) OvsNb
aroma
0.009 0.002 0.017
